The present invention is concerned with immunomodulatory and antimicrobial materials of invertebrate origin. In particular, the present invention is concerned with a composition, comprising a peptide or a peptide mixture of invertebrate origin and pharmaceutical preparations comprising such composition which are useful in the treatment of immune deficient conditions, infections and oncological diseases.
In the state of the art various pharmaceutical preparations of natural origin containing extractive materials of animal and plant tissues able to stimulate the immune system""s efficacy as well as to kill pathogenic microorganisms directly are known.
A process for obtaining cellular protein having anti-HIV activity from CD4-positive T cells or myeloid cells is disclosed in U.S. Pat. No. 5,480,782.
A topic formulation comprising a Ginkgo biloba extract exhibiting antibacterial and antiviral properties is disclosed in DE 43 34 600 A1.
WO 96/04005 discloses a pharmaceutical composition for stimulation of the immune response of an organism comprising as the active ingredient major histocompatibility complex antigens extracted from animal tissues, serum or cells. The tissues, cells or sera are chosen from goat, veal or pig liver and bovine red blood cells.
A pharmaceutical composition containing an extract of the plant Nigella sativa is disclosed in U.S. Pat. No. 5,482,711 for treating cancer, preventing the side effects of anticancer chemotherapy, and for increasing the immune functions in humans.
It is known from scientific literature that insect tissues also contain antimicrobial proteins and peptides (Gillespie J. P., Kanost M. R. and Trenczek T. Biological mediators of insect immunity. Annu. Rev. Entomol., 1997, 42, 611-643; Hoffmann J. A. and Reichhart J-M. Drosophila immunity Trends in Cell Biology, 1997, 7, 309-316). These materials posses direct toxicity to bacteria and fungi.
The preparations mentioned above and analogous natural pharmaceutical preparations enhance the recent arsenal of medicines suitable for treatment of immune deficient conditions, infections and oncological diseases. However, the pharmaceuticals which are available up to now do not cover existing demands in immunomodulatory and antibiotic medicines.
Therefore, it is an object of the present invention to make available a pharmaceutical composition useful for treatment of immune deficient conditions, infections and oncological diseases.
It has been found that compositions comprising a peptide or a peptide mixture of invertebrate and in particular insect origin exhibit an immunomodulatory and antimicrobial activity. Therefore, the present invention is related to a composition, comprising a peptide or a peptide mixture, preparable by a process comprising the steps of collecting, centrifugating and chromatographically separating the body fluid of an invertebrate.
Moreover, the present invention is directed to pharmaceutical preparations, comprising the above composition.
The compositions of the present invention comprise a peptide or a peptide mixture. Herein, the term xe2x80x9cpeptidexe2x80x9d refers to oligo- and polypeptides as well as to proteins. The peptides are believed to be the active principles in the pharmaceutical preparations prepared from these compositions. Nevertheless, the compositions may comprise further components which however should not interfere with the active principles in order to obtain useful pharmaceutical preparations.
The compositions of the present invention are preparable by a process comprising the steps of collecting, centrifugating and separating the body fluid of an invertebrate. The separation step can for example be carried out using a chromatographic column like a Sep-Pak C18 chromatographic column available from Waters Co., but other separation methods may also be used. If desired, further purification and concentration steps like lyophilisation may follow.
As source for the body fluid used for the isolation of the compositions of the present invention the following invertebrates have been found to be especially useful in the course of comparative investigations:
Calliphora vicina, Musca domestica, Limnephilus stigma, Sialis lutaria, Isogenus nubecula, Palomena prasina, Podisus maculiventris, Pachyiulus flaviceps, Hemophisalis longicornis, Arenicola marina, Mitilus edulis, Mya arenaria, Asterias rubens, Stychopus japonicus, Halocynthia roretzi and Aurelia aurita. 
Preferably the hemolymph of an insect may be used as the body fluid of an invertebrate. The hemolymph of an insect Calliphora vicina Robineau-Desvoidy (C. vicina, Diptera, Calliphoridae) is especially preferred.
It should be understood that the compositions of the present invention may be obtained by extraction from the body fluid of a single invertebrate as well as from the body fluids of two or more different invertebrates.
Moreover, it has been found that the hemolymph of septically injured C. vicina larvae accumulates unusual high concentrations of antimicrobial materials as compared to other species investigated. This allows to use C. vicina hemolymph as raw material for the production of pharmaceutical preparations with combinative antibiotic and immunomodulatory activity.
The C. vicina larvae may for example be septically injured by pricking off cuticle with a needle soaked in a suspension of heat-killed Escherichia coli and Micrococcus luteus cells. It is of course also possible to use other pathogenic or nonpathogenic bacteriae like Staphylococcus aureus or different strains of Salmonella.
Another aspect of the present invention are pharmaceutical preparations comprising one or more of the above compositions.
These pharmaceutical preparations are useful as antibiotics. Due to their antibiotic activity the pharmaceutical preparations of the present invention directly kill pathogenic microorganisms thereby exhibiting antibacterial and/or anti-fungal activity.
In vitro studies showed that the pharmaceutical preparations of the present invention have strong antibiotic activity against a variety of Gram-positive and Gram-negative bacteria including important human pathogens. The pharmaceutical preparations are also found to be effective in vivo. Particularly, they are able to cure mice infected with a lethal dose of human pathogenic strains of Klebsiella pneumonia and Salmonella typhimurium. Their fungicidal activity includes both yeast like Candida albicans and filamentous fungi.
Moreover, the pharmaceutical preparations of the present invention possess an immunomodulatory activity on human and mammalian immune cells and are thus useful for the treatment or prevention of immune deficient conditions like oncological diseases, especially cancer, and viral infections.
The basic mode of the immunomodulatory action of the pharmaceutical preparations of the present invention is a stimulation of cytotoxic lymphocytes such as natural killer cells able to kill malignant or virus-infected cells. The pharmaceutical preparations for example enforce mouse spleen lymphocytes to attack cancer cells of various origin in vitro and in vivo. Furthermore, the preparations when injected to mice induce strong and prolonged synthesis of endogenous interferon, the principal cytokin activating various defense mechanisms of the immune system.
Extensive studies of the response of lymphocytes of human donor blood to the pharmaceutical preparations of the present invention confirm that the pharmaceutical preparations also have a strong immunomodulatory effect on a significant part of the human population.
It is known that mechanisms of natural cytotoxicity play an important role in the organism""s protection against infection diseases as well as in the killing of the organism""s own malignant cells (Trinchieri G. Biology of natural killer cells, Advances in Immunology, 1989, vol. 47, 187-375; Brittenden J., Heys S. D., Ross J. and Eremin O. Natural killer cells and cancer, Cancer, 1996, vol. 77, 1126-1243). Therefore, stimulators of the lymphocytes"" natural cytotoxicity potentially may be used for the treatment and prevention of various infectious and cancer diseases caused by an insufficient efficacy of the cytotoxic mechanisms of innate immunity.
As for interferons, interleucins and other known immunomoludatory materials, the efficacy of the pharmaceutical preparations of the present invention is restricted by the basic capacity of the immune system to recognize and attack cancer cells. However, the efficacy may be enhanced when the pharmaceutical preparations are combined with other antitumor drugs. It has surprisingly been found that the pharmaceutical preparations of the present invention interact with different cytostatics and interferon and that some biocompatible combinations are active in cases when neither the known drug nor the pharmaceutical preparation is effective alone. Especially a combination of the pharmaceutical preparations of the present invention and bleomycin essentially suppressed tumor growth and increased life span even though each drug alone was much less effective. This synergistic effect between present pharmaceutical preparations and the known cytostatic bleomycin renders a combinative cancer immunochemotherapy possible which promises to be a most effective way to cancer treatment.
Instead of the compositions obtained from the body fluid of invertebrates the isolated or synthesized active principles of these compositions may be used for the preparation of the pharmaceutical preparations of the present invention. An active immunomodulatory principle of the composition of the present invention has been isolated. Preliminary data on the principle""s activity show that it is able to stimulate the cytotoxic anticancer activity of human and mouse lymphocytes in vitro as well as to induce in mice the lymphocytes"" cytotoxicity and the interferon production in vivo. A unique peculiarity of the active principle""s mode of action is its ability to stimulate the activity of the lymphocytes at extreme low concentrations. The minimum effective concentration was determined to be about 0.0005 nanogram/ml. The optimum concentration was found to be 0.05-0.5 nanogram/ml. In that case it stimulates the cytotoxicity of lymphocytes more effectively than interferon, a specific human cytokin responsible for the activation of lymphocytes.
A comparative analysis of the minimum active concentrations of an active principle of a composition of the present invention and the known human regulatory peptides interleucin, interferon, tumor necrosis factor, defensin and NK-lysin shows a clear advantage of the active principle. It works at an essentially less concentration as compared to any human cytokin including interleucin, the most active immunomodulator known so far. For the human cytolitic peptides NK-lysin and defensin which directly kill the target cells their concentration to enhance cancer cell destruction even exceeds the effective concentration of the active principle more than a million times.
Moreover, the compositions of the present invention and their active principles are effective stimulants of cytotoxic lymphocytes like NK-cells and cytotoxic T-cell and demonstrate potent antiviral activity when tested using as a model mice infected by human influenza virus A or B. In this model wild type males were infected intranasally by a suspension of the human influenza virus A or B and the pharmaceutical preparations were injected intraperitoneally one day before infection and then 1, 2, 4, 6 and 8 days after. Both, a composition of the present invention and its active principle effectively protected mice from pulmonary lesions and death. Thus, the composition and its active principle are useful in the preparation of a pharmaceutical preparation for the treatment or prevention of viral infections.
Neither an acute nor a chronic toxicity of the composition of the present invention or its active principle was found in the course of in vivo and in vitro studies.
It is understood that the pharmaceutical preparations of the present invention may also comprise conventional additives like excipients or carriers. The preparations may be administered to the patient by enteral, such as oral or rectal, and parenteral, such as intraperitoneal, intramuscular, intravenous or subcutaneous route. The preparations may be administered in dosage forms such as capsules, tablets and suppositories. For parenteral use the pharmaceutically active components are preferably in the form of an injectable solution.
The invention is further illustrated by the following examples: